ABSTRACT Human prion diseases are infectious and invariably fatal neurodegenerative diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, and variant CJD (vCJD) which is associated to consumption of cattle meat infected by bovine spongiform encephalopathy. Currently there is not sensitive, objective and non-invasive biochemical diagnosis for these diseases, and even less a procedure to detect people incubating the disease in the pre-symptomatic period. This is a major problem for public health, because prion diseases are known to transmit iatrogenically between human-to-human and because due to the long pre- symptomatic stage of the disease?which may span five decades?the asymptomatic carriers far outnumber the clinically affected individuals. To make the situation even more complicated, the infectious agent responsible for these diseases, termed prion, is composed exclusively of a conformationally altered form of a naturally occurring protein, named PrPSc, which has the unique ability to infect individuals and propagate in the body without the need for genetic material. PrPSc is not only the infectious agent and the likely culprit of neurodegeneration, but also the best surrogate marker for the disease. The challenge is that its quantity is high only in the brain at late stages of the disease. However, compelling evidences indicate that PrPSc is present in minute amounts in various peripheral tissues and biological fluids, including blood. This proposal is a continuation of a previously funded phase I/II fast track STTR project (R42NS079060) whose major goal was to develop a blood- and cerebrospinal fluid (CSF)-based detection assay for PrPSc associated with sCJD and vCJD. Our strategy utilizes the protein misfolding cyclic amplification (PMCA) technique, a pioneering proprietary technology developed in Dr Soto's lab. PMCA enables highly efficient prion replication in the test tube by amplification of this process in vitro, and offers a great opportunity to detect very small quantities of prions. During the previous funding cycle we were highly successful to reach the main aims and milestones, including the substantial optimization and improvement of the technology for human samples and the detection of PrPSc in samples of blood and urine of patients affected by vCJD with sensitivity and specificity approaching 100%. Another goal of the phase II was to obtain approval for using PMCA for disease diagnosis. Currently, a variation of the PMCA assay is successfully under use by the USA CJD Surveillance Center based on Cleveland, OH to help the diagnosis of CJD and Amprion is receiving royalty payments for this application. However, given the small number of cases, the diagnosis market is not profitable at this time. Amprion regards the screening of the blood supply as the most relevant market for commercialization of the Company's technology. Based on the success achieved during the phase I and II of this project we engaged in extensive discussions with the UK National Institute for Biological Standards and Control (NIBSC), the governmental organism trusted with the responsibility to validate and approve prion detection techniques in the UK and the Food and Drug Administration (FDA) to define the regulatory pathway to get approval for detection of prions in human blood. As a result of these discussions we have agreed to a validation pathway defined by these agencies to obtain regulatory approval for the commercialization of a PMCA-based assay for detection of prions in human blood samples and started the phase I of this process. The main goal of this Commercialization Readiness Pilot (CRP) project is to complete the regulatory and market approval of our test for detecting prions in blood of people infected by the vCJD agent and to increase the throughput of the assay to handle the number of samples expected during commercialization. The validation pathway includes 4 steps, namely: (i) testing a panel of blood samples spiked with vCJD brain or spleen extracts, (ii) a large screening of negative samples to assess specificity, (iii) a panel of samples from non-human primates infected with the vCJD agent and (iv) a group of samples from patients with confirmed vCJD. These samples will be provided blindly by the NIBSC or the FDA. The results generated in this project would lead to the development of a validated biochemical test to detect vCJD prions in human blood and the demonstration that this can be achieved before the clinical onset of the disease in asymptomatic carriers through the longitudinal studies in non-human primates experimentally infected with the vCJD agent. Availability of a validated methodology to screen blood for infectious prions will increase the safety of blood transfusions and potentially also products derived from human plasma. Finally, such a screening test could be utilized for a study of the size of the asymptomatic population of carriers (i.e. the number of people infected with the vCJD agent who have not yet developed the clinical disease). For commercialization of the validated test, Amprion will establish validated reference laboratories in different geographic locations (e.g. North America, Europe, Asia) that can handle the centralized testing of the samples with the accuracy, efficiency and safety needed for this type of assays.